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• W2084831435 abstract "A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4°C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4-fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups. A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4°C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4-fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups. Intracellular Ca2+ mobilization occurring in response to agonist stimulation of cells is mediated by the interaction of inositol 1,4,5-trisphosphate (IP3) 1The abbreviations and trivial names used are: IP3myo-inositol 1,4,5-trisphosphateIP3RIP3 receptorHEDTAN-hydroxyethylethylenediaminetriacetic acidPMSFphenylmethylsulfonyl fluoridemersalylO-(3-hydroxymercuri-2-methoxypropyl)carbamylphenoxyacetatethimerosal[(O-carboxyphenyl)thio]ethylmercury. with a specific receptor/Ca2+ channel(1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6186) Google Scholar). At least three different receptor isoforms have been identified by molecular cloning(2Mikoshiba K. Trends Pharmacol. Sci. 1993; 14: 86-89Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 3Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (320) Google Scholar, 4Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 5Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar). A domain model of the receptor has been proposed in which binding of IP3 to the N-terminal region of the receptor initiates a conformational change in the protein that gates a Ca2+ channel comprising six-transmembrane domains located in the C-terminal region(6Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (274) Google Scholar, 7Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Abstract Full Text PDF PubMed Google Scholar). It has been directly demonstrated that the purified IP3R is a functional Ca2+ channel(8Ferris C.D. Huganir R.L. Supattapone S. Snyder S.H. Nature. 1989; 342: 87-89Crossref PubMed Scopus (367) Google Scholar, 9Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Abstract Full Text PDF PubMed Google Scholar). myo-inositol 1,4,5-trisphosphate IP3 receptor N-hydroxyethylethylenediaminetriacetic acid phenylmethylsulfonyl fluoride O-(3-hydroxymercuri-2-methoxypropyl)carbamylphenoxyacetate [(O-carboxyphenyl)thio]ethylmercury. Ca2+ transients in single cells stimulated with suboptimal concentrations of agonists occur as repetitive spikes(1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6186) Google Scholar, 10Rooney T. Thomas A.P. Cell Calcium. 1993; 14: 674-690Crossref PubMed Scopus (79) Google Scholar). A number of models have been proposed to explain the complex behavior of Ca2+ signals recorded from individual cells. Experimental evidence suggests that the feed-back regulation of the IP3R plays a central role in the initiation and propagation of Ca2+ spikes. Small elevations of Ca2+ above resting levels have been shown to enhance IP3-mediated Ca2+ release(11Iino M. Endo M. Nature. 1992; 360: 76-78Crossref PubMed Scopus (244) Google Scholar, 12Finch E.A. Turner T.J. Goldin S.M. Science. 1991; 252: 443-446Crossref PubMed Scopus (676) Google Scholar, 13Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1441) Google Scholar). and Ca2+ sensitization of the IP3R to endogenous levels of IP3 has been proposed as one mechanism of Ca2+ wave propagation (14Miyazaki S. Yuzaki M. Nakada K. Shirakawa H. Nakanishi S. Nakade S. Mikoshiba K. Science. 1992; 257: 251-255Crossref PubMed Scopus (449) Google Scholar, 15Miyazaki S. Shirakawa H. Nakada K. Honda Y. Yuzaki M. Nkade S. Mikoshiba K. FEBS Lett. 1992; 309: 180-184Crossref PubMed Scopus (88) Google Scholar). The thiol-reactive agents t-butylhydroperoxide and thimerosal have also been shown to promote repetitive Ca2+ spiking in several cell types(15Miyazaki S. Shirakawa H. Nakada K. Honda Y. Yuzaki M. Nkade S. Mikoshiba K. FEBS Lett. 1992; 309: 180-184Crossref PubMed Scopus (88) Google Scholar, 16Bootman M.D. Taylor C.W. Berridge M.J. J. Biol. Chem. 1992; 267: 25113-25119Abstract Full Text PDF PubMed Google Scholar, 17Rooney T.A. Renard D.C. Sass E.J. Thomas A.P. J. Biol. Chem. 1991; 266: 12272-12282Abstract Full Text PDF PubMed Google Scholar, 18Bird G. Burgess G. Putney Jr., J.W. J. Biol. Chem. 1993; 268: 17917-17923Abstract Full Text PDF PubMed Google Scholar). In both instances, it is believed that the effects of these agents are related to a sensitization of the IP3R to endogenous levels of IP3. In the case of t-butylhydroperoxide, enhanced levels of oxidized glutathione (GSSG) are thought to underlie the sensitization. GSSG has been shown to decrease the half-maximal concentration of IP3 required for Ca2+ release from permeabilized hepatocytes(19Renard D.C. Seitz M.B. Thomas A.P. Biochem. J. 1992; 284: 507-512Crossref PubMed Scopus (74) Google Scholar). Low concentrations of thimerosal have also been shown to potentiate IP3-mediated Ca2+ release in several experimental systems(20Hilly M. Pietri-Rouxel F. Coquil J.F. Guy M. Mauger J.P. J. Biol. Chem. 1993; 268: 16488-16494Abstract Full Text PDF PubMed Google Scholar, 21Poitras M. Bernier S. Servant M. Richard D.E. Boulay G. Guillemette G. J. Biol. Chem. 1993; 268: 24078-24082Abstract Full Text PDF PubMed Google Scholar, 22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar, 23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar, 24Tanaka Y. Tashjian Jr., A. J. Biol. Chem. 1994; 269: 11247-11253Abstract Full Text PDF PubMed Google Scholar). The exact mode of action of thiol reagents on the IP3R has not been delineated, and it is not known if all of these agents have a common site of action. We have attempted to address this question by comparing the effects of several different thiol-reactive agents on [3H]IP3 binding and IP3-gated channel activity. In the present study we report that mersalyl and thimerosal, two structurally related organomercurial thiol-reactive agents, have different effects on the function of the isoforms of the IP3R in brain and liver. The data suggest the presence of several distinct reactive thiols that are important in regulating IP3R function. Mersalyl, thimerosal, N-ethylmaleimide, p-chloromercurophenylsulfonate were from Sigma. GSSG was from Boehringer Mannheim. Unlabeled IP3 was from Calbiochem. [3H]IP3 was from DuPont NEN. Isolated hepatocytes were prepared by collagenase digestion of perfused rat livers and were washed and stored on ice at 20-30 mg of protein/ml in Ca2+/Mg2+-free Hank's buffer as described previously(25Joseph S.K. Coll K.E. Thomas A.P. Rubin R. Williamson J.R. J. Biol. Chem. 1985; 260: 12508-12515Abstract Full Text PDF PubMed Google Scholar). For incubation with thiol-reactive agents, the cells were centrifuged (150 × g, 15 s) and resuspended in hepatocyte resuspension buffer which contained 120 mM KCl, 20 mM Tris-Hepes (pH 7.2), 2 mM HEDTA, 0.1 mM vanadate, 5 mM NaF, 4 nM okadaic acid, 1 mM PMSF, and 10 μg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor. The cells were permeabilized by addition of 40 μg of saponin/mg of cell protein. Complete permeabilization (<5 min) was monitored by trypan blue staining. The cell concentration was adjusted to 5 mg of protein/ml and 0.8 ml were incubated for 5 min at 4°C with 0.8 ml of label medium containing 120 mM KCl, 20 mM Tris-Hepes (pH 7.2), 10 nM [3H]IP3 (DuPont NEN; 20 Ci/mmol) in the presence or absence of thiol-reactive agents. Triplicate 0.5-ml samples were vacuum filtered through glass-fiber filters (Gelman A/E), and the filters were washed twice with 5 ml of 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, and 1 mg/ml bovine serum albumin. The filters were counted in scintillation fluid (Budget Solve, RPI Corp., Mount Prospect, IL). Permeabilized hepatocytes were solubilized at 4°C for 30 min in hepatocyte solubilization buffer, which contained 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (w/v) Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 μg/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin. Insoluble material was removed by centrifugation for 10 min at 25,000 × g. Binding to hepatocyte extracts was measured using a polyethylene glycol precipitation assay as described(26Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar). Intact hepatocytes (4 mg of protein/ml) were loaded with 5 μM Fura-2/AM for 35 min at 37°C in a buffer (pH 7.4) containing 10 mM Na+/Hepes, 120 mM NaCl, 4.7 mM KCl, 5 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 2% bovine serum albumin. After loading, the cells were washed and stored at 4°C in Na+/Hepes buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 20 mM Tris/Hepes, 1 mM KH2PO4, and 0.2 mM MgCl2. Prior to use, the cells were washed once in Na+/Hepes buffer containing 100 μM EGTA. The cells were then permeabilized at 4°C in a buffer (pH 7.2) containing 120 mM KCl, 10 mM NaCl, 20 mM Tris/Hepes, 1 mM KH2PO4, 0.2 mM MgCl2, 40 μg/ml digitonin, 5 μM carbonyl cyanide m-chlorophenylhydrazone, 5 μM oligomycin, 1 μM rotenone, 2 mM ATP, 5 mM phosphocreatine, 0.5 unit/ml creatine kinase, 1 μg/ml each pepstatin, antipain, and leupeptin, and 2 μM thapsigargin. Incubations were performed in the cuvette of a fluorimeter (Photon Technology Deltascan) maintained at 4°C with continuous stirring. The excitation wavelength was 360 nM with emission at 510 nM. The quenching of the Fura-2 trapped into the organelles was monitored after addition of 40 μM MnCl2. WB cells, a clonal cell line derived from rat liver(27Tsao M.S. Smith J.D. Nelson K.G. Grisham J.W. Exp. Cell Res. 1984; 154: 38-52Crossref PubMed Scopus (362) Google Scholar), were cultured in Richter's modified MEM containing 5% fetal bovine serum. Cells were grown in 100-mm dishes to confluence and were used between passages 25 and 30. The plates were washed twice in ice-cold phosphate-buffered saline and incubated at 4°C for 20 min in 1 ml of hypotonic buffer containing 10 mM Tris (pH 7.2), 1 mM EDTA, 0.2 mM PMSF, and 1 μg/ml pepstatin, antipain, and leupeptin. The cells from all the plates were scraped, pooled, and homogenized 15 times in a Dounce homogenizer with a tight fitting pestle. The homogenate was centrifuged at 100 × g for 1 min to remove unbroken cells. The supernatant was spun at 100,000 × g for 30 min. The crude WB cell membrane fraction was resuspended in 320 mM sucrose, 5 mM Tris-HCl (pH 7.8), 1 mM EDTA and stored at −80°C. Microsomal membranes were prepared from rat cerebellum homogenates by differential centrifugation as described previously (28Joseph S.K. Rice H.L. Williamson J.R. Biochem. J. 1989; 258: 261-265Crossref PubMed Scopus (94) Google Scholar) and stored at −80°C in the same buffer as WB cell membranes. WB cell membranes or rat cerebellum microsomal membranes were incubated at 1 mg of protein/ml in buffer A containing 120 mM KCl, 20 mM Tris-HCl (pH 7.4), and 1 mM EDTA in the presence and absence of 200 μM mersalyl for 10 min on ice. The membranes were pelleted by centrifugation at 100,000 × g for 30 min and resuspended in buffer A at 1 mg of protein/ml. Proteinase K was added at a ratio of 10 μg/mg membrane protein and incubated at room temperature for different periods of time. The reaction was terminated by addition of 3 mM PMSF and the membranes reisolated by centrifugation at 100,000 × g. The membrane pellet was solubilized and denatured in SDS-PAGE sample buffer. Samples were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose. The nitrocellulose sheets were immunoblotted with antibodies raised to amino acids 401-414 in the N-terminal domain (KEEK-Ab), or to the C-terminal 19 amino acids of the rat type-I IP3R. The recognition properties of the antibodies and conditions for immunoblotting have been previously described(29Mignery G.A. Sudhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (395) Google Scholar, 30Joseph S. Samanta S. J. Biol. Chem. 1993; 268: 6477-6486Abstract Full Text PDF PubMed Google Scholar, 31Joseph S.K. J. Biol. Chem. 1994; 269: 5673-5679Abstract Full Text PDF PubMed Google Scholar). Immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Amersham Corp.) The effect of several thiol-reactive agents on [3H]IP3 binding to saponin-permeabilized hepatocytes is shown in Fig. 1. The incubation period with thiol reagent and [3H]IP3 was 5 min and was performed at 4°C in a Mg2+-free medium to minimize hydrolysis of [3H]IP3. Under these conditions, a marked stimulation (4-5-fold) of [3H]IP3 binding was observed in the presence of mersalyl, an organomercurial thiol-reactive agent. Two structurally related molecules, thimerosal and p-chloromercurophenylsulfonate, also stimulated [3H]IP3 binding, but the degree of stimulation was much lower than observed with mersalyl. Increasing the concentration or incubation time did not enhance the stimulatory effect of thimerosal (data not shown). Oxidized glutathione or the thiol-alkylating agent, N-ethylmaleimide, were without effect on [3H]IP3 binding at 4°C. Ca2+ has been shown to stimulate IP3 binding to the hepatic IP3R(32Mauger J.P. Claret M. Pietri F. Hilly M. J. Biol. Chem. 1989; 264: 8821-8826Abstract Full Text PDF PubMed Google Scholar). It has been proposed that Ca2+ mediates a conversion of receptors from a low affinity (active) form to a high affinity (inactive) form(33Pietri F. Hilly M. Mauger J.P. J. Biol. Chem. 1990; 265: 17478-17485Abstract Full Text PDF PubMed Google Scholar, 34Rouxel F.P. Hilly M. Mauger J.P. J. Biol. Chem. 1992; 267: 20017-20023Abstract Full Text PDF PubMed Google Scholar). Hilly et al.(20Hilly M. Pietri-Rouxel F. Coquil J.F. Guy M. Mauger J.P. J. Biol. Chem. 1993; 268: 16488-16494Abstract Full Text PDF PubMed Google Scholar) have shown previously that the stimulatory effect of Ca2+ and thimerosal on IP3 binding to permeabilized hepatocytes are not additive. Fig. 2A demonstrates that the stimulation of [3H]IP3 binding by mersalyl is dose-dependent with maximal effects being observed at 100-200 μM mersalyl. Ca2+ (buffered at 10 μM concentration) stimulated IP3 binding to a lesser extent than a maximal concentration of mersalyl and the effect of both agents were not additive. Scatchard analysis of the binding data (Fig. 2B), indicated that mersalyl stimulated [3H]IP3 binding by increasing the affinity of the IP3 receptor without altering the maximal number of binding sites. In three experiments the respective apparent Kd and Bmax values were 27 ± 3 nM and 125 ± 6 fmol/mg protein under control conditions, and 4.7 ± 0.3 nM and 98 ± 3 fmol/mg protein in the presence of 100 μM mersalyl. The effect of mersalyl is qualitatively similar to the effect of Ca2+ on the binding affinity of the hepatic IP3R(33Pietri F. Hilly M. Mauger J.P. J. Biol. Chem. 1990; 265: 17478-17485Abstract Full Text PDF PubMed Google Scholar). We have shown previously that the stimulatory effect of Ca2+ on IP3 binding in permeabilized hepatocytes is lost after detergent solubilization of membranes(26Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar). The experiment in Fig. 3 was carried out to determine if this was also the case with mersalyl. Permeabilized hepatocytes were treated with mersalyl and then washed. The washed mersalyl-treated hepatocytes retained an enhanced IP3 binding activity (Fig. 3A). These hepatocytes were then solubilized with Triton X-100, and binding activity was measured in extracts exposed to increasing concentrations of mersalyl (Fig. 3B). The addition of mersalyl to Triton X-100 extracts prepared from control hepatocytes had no significant effect on [3H]IP3 binding. Binding activity in Triton X-100 extracts of mersalyl pretreated membranes was only slightly higher than control extracts and the further addition of mersalyl produced a dose-dependent inhibition of binding. These results indicate that, as with Ca2+, the stimulatory effect of mersalyl cannot be observed after detergent solubilization. The reason why mersalyl pretreatment of membranes causes mersalyl to inhibit [3H]IP3 binding in detergent extracts is presently not clear. A possibility is that mersalyl binding to the receptor in membranes alters the conformation of the protein (see below) in a manner that exposes an additional mersalyl-reactive thiol group after detergent solubilization that is inhibitory to ligand binding. We have examined the functional effect of mersalyl and thimerosal on IP3-mediated Ca2+ channel function in Figure 4:, Figure 5:. In order to avoid the known inhibitory effect of thiol-reactive agents on Ca2+-ATPase (16Bootman M.D. Taylor C.W. Berridge M.J. J. Biol. Chem. 1992; 267: 25113-25119Abstract Full Text PDF PubMed Google Scholar, 22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar, 35Missiaen L. Taylor C.W. Berridge M.J. Nature. 1991; 352: 241-244Crossref PubMed Scopus (275) Google Scholar), we have utilized an assay method based on the ability of Mn2+ to traverse the IP3-activated Ca2+ channel in a retrograde manner and quench the fluorescence of Fura-2 compartmentalized in intracellular stores(36Renard-Rooney D.C. Hajnoczky G. Seitz M.B. Schneider T. Thomas A.P. J. Biol. Chem. 1993; 268: 23601-23610Abstract Full Text PDF PubMed Google Scholar). All the fluorescence measurements were carried out at 4°C in order to permit comparison to the ligand binding data. The addition of Mn2+ to permeabilized Fura-2-loaded hepatocytes, pretreated with the Ca2+ pump inhibitor thapsigargin, produced a rapid quenching of cytosolic Fura-2 released from the permeabilized cells. The subsequent addition of 1 μM IP3 (a maximal dose) produced an additional quench corresponding to the entry of Mn2+ into the IP3-sensitive compartment (Fig. 4). Further entry of Mn2+ into the IP3-insensitive compartment could be observed after addition of ionomycin. When the permeabilized hepatocytes were pretreated with mersalyl a complete inhibition of the IP3-mediated quench was observed (Fig. 4). The total pool size of the intracellular stores was not altered by mersalyl pretreatment. Greater than 95% inhibition of IP3-mediated Mn2+ quench was observed with IP3 concentrations in the range 0.1-10 μM (data not shown). The inhibitory effects of mersalyl were also noted at 37°C, although the effects were less marked than at 4°C. 2The initial rate of IP3 (1 μM) induced Fura-2 quenching at 37°C expressed as percentage of total fluorescence/s was 2.02 ± 0.35 in control cells and 0.35 ± 0.07 in mersalyl-treated cells. Under these conditions, the total quench mediated by IP3 expressed as a percentage of the ionomycin quenchable pool was 37.5 ± 2.7% in control cells and 19.4 ± 3.4% in mersalyl-treated cells (mean ± S.E., n = 4). Previous studies have shown that the potentiating effect of thimerosal on IP3-induced Ca2+ release is seen only at suboptimal IP3 concentrations(22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar, 23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar). In agreement with these studies, thimerosal at 4°C markedly potentiated the Mn2+ quench mediated by 10 nM IP3 (Fig. 5, lower panel) with a much smaller effect on the responsiveness to 500 nM IP3 (Fig. 5, upper panel).Figure 5:The effect of thimerosal on IP3-mediated Mn2+ quenching of compartmentalized Fura-2 in intracellular stores. Hepatocytes were loaded with Fura-2/AM, washed, and permeabilized at 4°C as described under “Experimental Procedures.” The permeabilized cells were pretreated with thimerosal (100 μM) for 5 min before addition of Mn2+. All recordings were made at 4°C.View Large Image Figure ViewerDownload (PPT) A possible mechanism of action of mersalyl is that binding of this agent to a free thiol group on the IP3R alters the conformation of the protein, resulting in a form of the receptor with an inactive Ca2+ channel and a high affinity for ligand. To try to detect a conformational change in the protein in its native membrane environment, we have looked for changes in the pattern of immunoreactive fragments generated after addition of proteases. Such experiments are facilitated by using membranes that contain higher levels of immunoreactive IP3R than found in hepatocytes. Fig. 6 shows the effect of mersalyl on [3H]IP3 binding to membranes prepared from WB rat liver epithelial cells and rat cerebellum. Both membranes are known to contain relatively high levels of immunoreactive type-I IP3R(31Joseph S.K. J. Biol. Chem. 1994; 269: 5673-5679Abstract Full Text PDF PubMed Google Scholar, 37Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (826) Google Scholar). Only the receptor in WB membranes showed a stimulation of [3H]IP3 binding by mersalyl, and these were used in subsequent studies. Fig. 7A shows the pattern of fragments observed after proteinase K digestion of WB cell membranes, as visualized with an antibody raised to amino acids 401-414 in the N-terminal region of the type-I IP3R. In addition to several intermediate digestion products, a prominent proteolytic product of 37.1 ± 2.2 kDa (n = 4) was formed with progressive proteinase K digestion under control conditions. This band was not observed after proteinase K treatment of WB cell membranes that had first been treated with mersalyl and reisolated by centrifugation. The protease fragmentation pattern of cerebellum IP3R was substantially different from that observed with WB cell membranes, although a 35.4 ± 0.4 kDa (n = 3) polypeptide was also formed in cerebellar membranes (Fig. 7B). In agreement with the results of binding data, pretreatment of cerebellum membranes with mersalyl had no effect on the protease cleavage pattern. Fig. 8A shows that thimerosal, at an equivalent concentration, does not mimic the action of mersalyl. The effect of mersalyl on the protease cleavage pattern of the WB IP3R was completely prevented by inclusion of an excess of dithiothreitol. The 37-kDa immunoreactive fragment in both WB cell and cerebellum was associated with the membrane fraction. This reflected a peripheral association with the membrane, since we were able to remove this fragment by washing the membranes with 0.1 M Na2CO3, pH 11.0 (data not shown). The supernatant fractions obtained after proteinase K digestion of control and mersalyl-treated WB membranes were analyzed for the presence of the 37-kDa KEEK-reactive fragment (Fig. 8B). The absence of the 37-kDa cleavage product in WB membranes after mersalyl pretreatment was not the result of a selective loss of this fragment into the soluble fraction. The combination of mersalyl and thimerosal together produces a pattern of protease digestion which is the same as obtained with mersalyl alone (Fig. 8C). This observation supports the conclusion that the two sulfydryl reagents interact with different thiol groups. Several recent studies have examined the effect of thimerosal on IP3 binding and IP3-dependent Ca2+ release. Low concentrations of thimerosal were found to potentiate IP3-mediated Ca2+ release from adrenal cortex microsomes(21Poitras M. Bernier S. Servant M. Richard D.E. Boulay G. Guillemette G. J. Biol. Chem. 1993; 268: 24078-24082Abstract Full Text PDF PubMed Google Scholar), permeabilized hepatocytes(20Hilly M. Pietri-Rouxel F. Coquil J.F. Guy M. Mauger J.P. J. Biol. Chem. 1993; 268: 16488-16494Abstract Full Text PDF PubMed Google Scholar), cerebellum microsomes (20Hilly M. Pietri-Rouxel F. Coquil J.F. Guy M. Mauger J.P. J. Biol. Chem. 1993; 268: 16488-16494Abstract Full Text PDF PubMed Google Scholar, 22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar), and permeabilized A7r5 smooth-muscle cells(23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar). In adrenal cortex and hepatocytes, the potentiation by thimerosal was accompanied by a large stimulation of IP3 binding reflecting an increased affinity of the IP3R. On the basis of these data, it has been concluded that the high affinity form of the receptor induced by thimerosal is functionally active and is distinct from the high affinity form of the receptor induced by Ca2+, which is functionally less active as a Ca2+ channel(33Pietri F. Hilly M. Mauger J.P. J. Biol. Chem. 1990; 265: 17478-17485Abstract Full Text PDF PubMed Google Scholar). Our data show that the related thiol-reagent, mersalyl, has a marked stimulatory effect on IP3 binding and behaves more like Ca2+, in that it generates a high affinity form of the receptor that is functionally inactive. These studies reinforce the idea that the IP3R can exist in several non-equivalent high affinity states that may have high or low Ca2+ conductance (20Hilly M. Pietri-Rouxel F. Coquil J.F. Guy M. Mauger J.P. J. Biol. Chem. 1993; 268: 16488-16494Abstract Full Text PDF PubMed Google Scholar, 38Marshall I.C.B. Taylor C.W. J. Biol. Chem. 1993; 268: 13214-13220Abstract Full Text PDF PubMed Google Scholar). It should be noted that the stimulatory effect of thimerosal on IP3 binding has not been observed in all studies (e.g.(22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar) and (23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar), and only a modest stimulation of binding was observed in permeabilized hepatocytes under our assay conditions at 4°C. We attribute this difference to reaction of the sulfydryl reagents with separate thiol groups on the receptor rather than to differences of reactivity with the same thiol group for several reasons. First, incubation of permeabilized hepatocytes at 4°C for longer periods with higher concentrations of thimerosal did not enhance the effect on [3H]IP3 binding. Second, the effects of mersalyl and thimerosal on channel function and protease digestion of the receptor are clearly different. Third, the combination of mersalyl and thimerosal together produces a pattern of protease digestion which is the same as obtained with mersalyl alone (Fig. 8C). Other factors, such as temperature, may modify the reactivity of individual thiol groups. For example, we have found that the effect of thimerosal on ligand binding could be enhanced, and the effect of mersalyl diminished, by preincubating the permeabilized hepatocytes at 37°C with the thiol agents prior to measurement of binding at 4°C. 3D. Renard-Rooney, S. K. Joseph, M. Seitz, and A. P. Thomas, unpublished observations. The elevation of temperature does not, however, qualitatively modify the effect of the sulfydryl agents on channel function (Footnote 2 and data not shown). A temperature-dependent alteration in the accessibility of N-ethylmaleimide-reactive thiol groups on the hepatic IP3R has also been documented previously(39Pruijn F.B. Sibeijn J.P. Bast A. Biochem. Pharmacol. 1990; 40: 1947-1952Crossref PubMed Scopus (21) Google Scholar). Whereas low concentrations of thimerosal stimulate IP3-mediated Ca2+ release, higher concentrations (>10 μM) have been found to inhibit the release process in some systems(22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar, 23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar). In part, this is due to an inhibition of the Ca2+ pump and to an increase in the passive Ca2+ permeability of the endoplasmic reticulum membrane(22Sayers L.G. Brown G.R. Michell R.H. Michelangeli F. Biochem. J. 1993; 289: 883-887Crossref PubMed Scopus (72) Google Scholar, 23Parys J. Missiaen L. De Smedt H. Droogmans G. Casteels R. Pflugers Arch. 1993; 424: 516-522Crossref PubMed Scopus (54) Google Scholar). Using the Mn2+ quench assay, a biphasic dependence on thimerosal concentrations up to 100 μM was not observed in the present study. Mersalyl stimulates IP3 binding to liver and WB cell membranes but is without effect on cerebellum microsomes. The full spectrum of IP3R isoforms in each of these tissues has not been established unequivocally. However, it is known that the cerebellum contains predominantly type-I IP3R (3Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (320) Google Scholar, 40Ross C.A. Danoff S.K. Schell M.J. Snyder S.H. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4265-4269Crossref PubMed Scopus (220) Google Scholar) and that non-neuronal cells contain an alternative transcript of the type-I isoform that has a 40-amino acid deletion(41Danoff S.K. Ferris C.D. Donath C. Fischer G.A. Munemitsu S. Ullrich A. Snyder S.H. Ross C.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2951-2955Crossref PubMed Scopus (215) Google Scholar, 42Nakagawa T. Okano H. Furuichi T. Aruga J. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6244-6248Crossref PubMed Scopus (207) Google Scholar). These “long” and “short” forms of the type-I IP3R have been shown to differ in their regulation by Ca2+ and cAMP-dependent phosphorylation(41Danoff S.K. Ferris C.D. Donath C. Fischer G.A. Munemitsu S. Ullrich A. Snyder S.H. Ross C.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2951-2955Crossref PubMed Scopus (215) Google Scholar). It has recently been reported that the liver expresses low amounts of type-III IP3R mRNA and much higher amounts of type-I and type-II IP3R mRNA(43De Smedt H. Missiaen L. Parys J.B. Bootman M.D. Mertens L. Van Den Bosch L. Casteels R. J. Biol. Chem. 1994; 269: 21691-21698Abstract Full Text PDF PubMed Google Scholar). By contrast, the WB cell contains predominantly types I and III IP3R and very little of any other isoform, as judged by the 90% immunodepletion of [3H]IP3 binding sites from WB cell extracts by a combination of type-I and type-III specific antibodies. 4S. K. Joseph, S. Pierson, and A. P. Maranto, unpublished observations. Thus the difference in reactivity toward mersalyl of the hepatocyte/WB cell and cerebellum IP3R could be accounted for by the selective insensitivity of the type-I (long form) to the sulfydryl agent or alternatively, to tissue-specific differences in ancillary sulfydryl reagent-sensitive regulatory proteins. It is somewhat surprising that two such closely related organomercurial agents should have different effects on the hepatic IP3R. Both molecules are anionic and, in the case of mersalyl, known not to penetrate mitochondrial membranes(44Fonyo A. J. Bioenerg. Biomembr. 1978; 10: 171-194Crossref PubMed Scopus (72) Google Scholar). The inhibitory effects of mersalyl are believed to be the result of the formation of a mercaptide bond with free thiol groups in a protein(45van Iwaarden P.R. Driessen A.J.M. Konings W.N. Biochim. Biophys. Acta. 1992; 1113: 161-170Crossref PubMed Scopus (107) Google Scholar). With thimerosal, the mercury is already linked to sulfur, and it is therefore possible that reaction with protein thiols may generate mixed disulfides. In studies on the rate of reaction of the free thiols of ovalbumin with several mercaptide forming agents, it was observed that mersalyl was more reactive than other agents such as p-chloromercurobenzoate (46Taketera K. Watanabe T. Anal. Chem. 1993; 65: 3644-3646Crossref PubMed Scopus (32) Google Scholar). These variations in reactivity were attributed to differences in the degree of steric hindrance around the reactive mercury in these molecules(46Taketera K. Watanabe T. Anal. Chem. 1993; 65: 3644-3646Crossref PubMed Scopus (32) Google Scholar). Therefore, differences in the chemistry of reaction and/or accessibility to reactive thiol groups may underlie the distinctive effects of the two agents. The current model of IP3 gating of the Ca2+ channel in the IP3R proposes that ligand binding is associated with a large conformational change in the protein(6Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (274) Google Scholar). In the present study, we have shown that mersalyl pretreatment of WB cell membranes effectively eliminates the appearance of a 37-kDa immunoreactive receptor fragment generated by proteinase K cleavage. We interpret these data to indicate that reaction of a thiol group on the receptor induced a conformational change in the protein that exposed the region of the receptor containing the antibody epitope (amino acids 401-414) to cleavage by proteinase K. Two findings suggest that this conformational change is linked to the effect of mersalyl on [3H]IP3 binding. First, mersalyl does not affect [3H]IP3 binding in cerebellum microsomes and also does not alter the formation of any of the proteolytic fragments in this system. Second, thimerosal does not mimic the effect of mersalyl on [3H]IP3 binding or the appearance of 37-kDa proteinase K fragment. The antibody epitope falls within the region thought to be involved in ligand binding(2Mikoshiba K. Trends Pharmacol. Sci. 1993; 14: 86-89Abstract Full Text PDF PubMed Scopus (137) Google Scholar). However, IP3 itself had no effect on the pattern of proteinase K digestion fragments and did not prevent the effect of mersalyl (data not shown). [3H]IP3 binding to Triton X-100 solubilized extracts (Fig. 3B) or heparin-agarose column eluates (data not shown) are not affected by mersalyl (data not shown). Mersalyl may continue to react with thiol groups on the receptor under these conditions and still have no effect on binding, since removal of the protein from its membrane environment may grossly alter regulation of the binding site(26Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar). However, the present data do not exclude the possibility that mersalyl or other sulfydryl agents interact with ancillary regulatory proteins rather than reacting directly with the receptor. Despite this, it is clear that reaction of specific thiol groups on the receptor, or a regulatory protein, has profound consequences on the function of the IP3 receptor/ion channel. These thiols have varied sensitivity to sulfydryl reagents, and reactivity is also different between different IP3R isoforms. Identification of these functionally important cysteine residues and characterization of their role in ligand binding/ion channel function of the receptor remains a challenging objective." @default.
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